Detection of DNA in specimens comprising body fluids or tissues can be difficult because of the small quantity of DNA present or because of the presence in the specimen of other interfering materials, including DNA from a different source. These limitations may be overcome by employing an analytic method referred to as the polymerase chain reaction (PCR) technique. By this technique, selective enrichment of a specific DNA sequence can be achieved by exponential amplification of the target sequence. Mullis, et al., Met. Enzymol., 155, 335 (1987).
To facilitate PCR amplification, pairs of oligonucleotide primers may be employed as described in U.S. Pat. No. 4,683,202 (hereby incorporated by reference). The primers are designated to hybridize with sequences that flank the target DNA. Following in vitro amplification, the amplified target sequence is detected by a hybridizing probe. For example, this analytical procedure has been used for the direct detection of HIV-1 as described by Ou, et al., Science, 238, 295-97 (1988). The amplification cycles are facilitated by using a polymerase which is thermally stable in incubations up to 95 degrees centigrade, as described by Saiki, et al., Science, 239, 487-91 (1988).
Bovine respiratory syncytial virus (BRSV), a pneumovirus in the family Parayxoviridae, is an important cause of acute respiratory disease in postweaning calves and feedlot cattle in the United States. In Europe, BRSV infection is considered to be one of the most significant causes of bovine respiratory disease. Although most infections are not apparent, the high prevalence of seropositive cattle in the United States indicated that infection rates are high.
A major problem with establishing a more accurate understanding of BRSV's role in bovine respiratory disease is the difficulty associated with rapidly and accurately identifying the presence of the virus. Successful laboratory diagnosis of BRSV is generally based on one of three criteria: (1) virus isolation, (2) identifying BRSV antigens in suspected tissue, or (3) indications of BRSV seroconversion. However, the lack of standardized reagents, the high prevalence of cattle with antibody titers to BRSV (60-80% seropositive) and the need for skilled personnel to process and interpret fluorescent antibody results have hindered development of routine diagnostic test. Similarly, successful virus isolations from typical clinical cases of BRSV infection are often unsuccessful and can take from 11-21 days because of the late appearance of any noticeable cytopathic effect. Because of these difficulties, direct isolation of BRSV is not recommended as a routine procedure. See Edwards et al., Respiratory Syncytial Virus Diagnosis, Vet. Res. 114:101 (1984).
Human respiratory syncytial (HRSV) is the most important cause of respiratory disease in infants and young children, and the rapid detection of HRSV antigens in clinical specimens has been useful in diagnosing and limiting the nosocomial spread of HRSV associated bronchiolitis and pneumonia. Similarly, synthetic oligonucleotide probes have been used for in situ hybridizations to identify HRSV in nasopharyngeal secretions (Cubie et. al. Detection of Respiratory Syncytial Virus Antigen and Nucleic Acid in Clinical Specimens using Synthetic Oligonucleotides, J. Virol. Methods. 34: 27-35 (1991), and cDNA probes and synthetic oligonucleotides have been used to differentiate HRSV subgroups (Sullender et al., Genetic Diversity of the Attachment Protein of Subgroup B Respiratory Syncytial Viruses, J. Viro. 65:5425-34 (1991), Sullender et al.,Synthetic Oligonucleotide Probes Differentiate Respiratory Syncytial Virus Subgroups in a Nucleic Acid Hybridization Assay, J. Clin. Microbiol. 29:1255-57 (1991). Additionally, reverse transcription and polymerase chain reactions have been combined to amplify and detect low levels of HRSV mRNA from nasopharyngeal aspirates (Paton et al., Rapid Detection of Respiratory Synbctial Virus in Nasopharyngeal Aspirates by Reverse Transcription and Polymerase Chain Reaction Amplification, J. Clin. Microbiol. 30:901-904 (1992) and otitis media effusion (Okamoto et al., Genetic Sequences of Respiratory Syncyntial Virus in Otitis Media with Effusion, Lancet 338;1025-26 (1992).
With this background in mind a method for amplifying, detecting and differentiating BRSV fusion (F) protein mRNA from HRSV mRNA using RT-PCR and oligonucleotides probes was developed.